Alexander S. Raikhel PROJECT SUMMARY/ABSTRACT The damaging effect of mosquito-borne diseases is colossal, with about a million deaths and hundreds of thousands of people becoming seriously ill annually. Being the principal vector for dengue, yellow fever, chikungunya virus and recently Zika virus, the Aedes aegypti mosquito is an extremely important organism for investigation. Small non-coding microRNAs (miRNA) are responsible for posttranscriptional control of mRNAs and act as essential regulators of development. Our efforts during the previous funding cycle have revealed the role of miRNAs in controlling multiple physiological functions in female mosquitoes. Moreover, we have established a number of essential methodologies for mosquito miRNA investigations, from bioinformatics and miRNA library sequencing to genetics approaches such as CRISPR/Cas9 and GAL4/UAS. These studies have provided a foundation for further exploration of mosquito miRNAs. However, our knowledge about miRNA roles in such an essential process as female mosquito reproduction is still limited. Thus, the goal of the present grant proposal is to elucidate the involvement of miRNAs in regulatory networks governing blood-meal-activated reproductive events in female mosquitoes. The overall hypothesis is that miRNAs are critical components of these regulatory networks. We will examine the following hypotheses in Specific Aims: Aim 1. miRNAs are involved in posttranscriptional regulation of enzymes essential for blood digestion. Aim 2. miRNAs are required for the control of key factors of transcriptional networks that are necessary for orchestrating the female mosquito reproductive cycle. Aim 3. As important regulatory molecules, miRNAs are under strict control, coordinating their activity with demands of mosquito reproduction. The innovation of this proposed research rests on a comprehensive analysis of the role of miRNAs in mosquito reproduction and application of wide-ranging methodologies, such as CRISPR-Cas9 and Gal4-UAS. The significance of the present grant proposal is in establishing a novel paradigm concerning the association of miRNAs with regulatory networks controlling mosquito reproduction.